Compositions and methods for inhibiting the interaction between CFTR and CAL

ABSTRACT

The present invention features compositions and methods for increasing the cell surface expression of degradation-prone CFTR proteins and preventing or treating cystic fibrosis. The invention provides peptides and peptidomimetics that selectively inhibit the interaction between CAL and mutant CFTR proteins, thereby stabilizing the CFTR and facilitating transport of the same to the cell surface.

This application is the National Stage of International Application No.PCT/US2009/061246 filed Oct. 20, 2009, which claims the benefit ofpriority of U.S. Provisional Application No. 61/107,438, filed Oct. 22,2008, the contents of each of which are incorporated herein by referencein their entirety. Work on this invention was also supported by grantsfrom the Cystic Fibrosis Foundation.

This invention was made with government support under Grant No.R01-DK075309 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) is the targetof mutations that cause cystic fibrosis (CF). CF is characterized byabnormal endocrine and exocrine gland function. In CF, unusually thickmucus leads to chronic pulmonary disease and respiratory infections,insufficient pancreatic and digestive function, and abnormallyconcentrated sweat. Seventy percent of the mutant CFTR alleles in theCaucasian population result from deletion of phenylalanine at position508 (ΔF508-CFTR), the result of a three base pair deletion in thegenetic code. Other mutations have also been described, e.g., a glycineto aspartate substitution at position 551 (G551D-CFTR) occurs inapproximately 1% of cystic fibrosis patients.

The ΔF508-CFTR mutation results in a CFTR protein capable of conductingchloride, but absent from the plasma membrane because of aberrantintracellular processing. Under usual conditions (37° C.), theΔF508-CFTR protein is retained in the endoplasmic reticulum (ER), byprolonged association with the ER chaperones, including calnexin andhsp70. Over expression of ΔF508-CFTR can result in ΔF508-CFTR proteinappearing at the cell surface, and this protein is functional once itreaches the cell surface. The ΔF508-CFTR “trafficking” block is alsoreversible by incubation of cultured CF epithelial cells at reducedtemperatures (25-27° C.). Lowered temperature results in the appearanceof CFTR protein and channel activity at the cell surface, suggesting anintrinsic thermodynamic instability in ΔF508-CFTR at 37° C. that leadsto recognition of the mutant protein by the ER quality controlmechanism, prevents further trafficking, and results in proteindegradation. Chemical chaperones are currently being developed torestore the folding of ΔF508-CFTR. However, when ΔF508-CFTR is expressedat the cell-surface following treatment, CAL (also known asCFTR-associated ligand, PIST, GOPC, ROS, and FIG) directs the lysosomaldegradation of CFTR in a dose-dependent fashion and reduces the amountof CFTR found at the cell surface. Conversely, NHERF1 and NHERF2functionally stabilize CFTR. Consistent with this role of CAL, RNAinterference targeting of endogenous CAL also increases cell-surfaceexpression of the disease-associated ΔF508-CFTR mutant and enhancestransepithelial chloride currents in a polarized human patient bronchialepithelial cell line (Wolde, et al. (2007) J. Biol. Chem.282:8099-8109).

Current treatments for cystic fibrosis generally focus on controllinginfection through antibiotic therapy and promoting mucus clearance byuse of postural drainage and chest percussion. However, even with suchtreatments, frequent hospitalization is often required as the diseaseprogresses. New therapies designed to increase chloride ion conductancein airway epithelial cells have been proposed, and restoration of theexpression of functional CFTR at the cell surface is considered a majortherapeutic goal in the treatment of cystic fibrosis, a disease thataffects ˜30,000 patients in the U.S., and ˜70,000 patients worldwide.Indeed, screening assays have been described for identifying agents thatmodify or restore cell surface expression of mutant CFTR proteins.However, only a limited number of “corrector” drugs has been describedfor the treatment of CF. In addition, U.S. Patent Application No.20050282743 discloses reagents and methods for inhibiting interactionsbetween proteins in cells, particularly interactions between a PDZprotein such as PIST and a PL protein such as wild-type CFTR. However,no high-affinity and selective inhibitor compounds have been identifiedfor PIST, nor have PIST reporter sequences been identified that wouldpermit small-molecule screening, nor have any such compounds been shownto have efficacy in stabilizing mutant, degradation-prone CFTR.Accordingly, improvements are needed in the treatment of cysticfibrosis. The present invention fulfills this need and further providesother related advantages.

SUMMARY OF THE INVENTION

The present invention features methods for increasing cell surfaceexpression of a degradation-prone Cystic Fibrosis TransmembraneConductance Regulator (CFTR) protein and a method of preventing ortreating CF in a subject in need of treatment. The methods of theinvention employ an agent that selectively inhibits the interactionbetween the degradation-prone CFTR and CFTR-Associated Ligand (CAL)thereby increasing cell surface expression of the degradation-prone CFTRprotein. In one embodiment, the degradation-prone CFTR is ΔF508 CFTR orR1066C CFTR. In other embodiments of the invention, the agent is apeptide or peptidomimetic of 6 to 20 residues in length. In certainembodiments, the peptide comprises the amino acid sequence of SEQ IDNO:1 or a derivative thereof. In particular embodiments, the peptide islisted in Table 1. In another embodiment, the peptidomimetic is amimetic of the amino acid sequence of SEQ ID NO:1. In a furtherembodiment, the peptidomimetic is listed in Table 2.

Peptides, derivatives, peptidomimetic, as well as compositionscontaining the same are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Novel inhibitors have now been identified that block the interaction orbinding of CFTR with the CAL PDZ binding site by competitivedisplacement. By inhibiting this interaction with CAL, degradation-proneCFTR proteins are stabilized and the amount of CFTR protein at the cellsurface is effectively increased. Indeed, representative peptide andpeptidomimetic CAL inhibitors were shown to increase the apicalcell-surface expression and transepithelial chloride efflux of the mostcommon CFTR mutation associated with CF. Accordingly, inhibitors of thepresent invention find application in increasing the cell surfaceexpression of degradation-prone CFTR proteins and in the treatment forCF. As used herein, “cell surface expression” of a CFTR protein refersto CFTR protein which has been transported to the surface of a cell. Inthis regard, an agent that increases the cell surface expression of aCFTR protein refers to an agent that increases the amount of CFTRprotein, which is present or detected at the plasma membrane of a cell,as compared to a cell which is not contacted with the agent.

Genetic, biochemical, and cell biological studies have revealed acomplex network of protein-protein interactions that are required forcorrect CFTR trafficking, including a number of PDZ (PSD-95,discs-large, zonula occludens-1) proteins, which act as adaptormolecules, coupling CFTR to other components of the trafficking andlocalization machinery, and to other transmembrane channels andreceptors (Kunzelmann (2001) News Physiol. Sci. 16:167-170; Guggino &Stanton (2006) Nat. Rev. Mol. Cell. Biol. 7:426-436). Class I PDZdomains typically recognize C-terminal binding motifs characterized bythe sequence—(Ser/Thr)-X-Φ-COOH (where Φ represents a hydrophobic sidechain, and X represents any amino acid) (Harris & Lim (2001) J. CellSci. 114:3219-3231; Brône & Eggermont (2005) Am. J. Physiol.288:C20-C29). The cytoplasmic C-terminus of CFTR satisfies the class IPDZ binding motif, ending in the sequence—Thr-Arg-Leu (Hall, et al.(1998) Proc. Natl. Acad. Sci. USA 95:8496-8501; Short, et al. (1998) J.Biol. Chem. 273:19797-19801; Wang, et al. (1998) FEBS Lett. 427:103-108)and it has been demonstrated that CFTR C-terminal PDZ-binding motifcontrols retention of the protein at the apical membrane and modulatesits endocytic recycling (Moyer, et al. (2000) J. Biol. Chem.275:27069-27074; Swiatecka-Urban, et al. (2002) J. Biol. Chem.277:40099-40105). PDZ proteins that have been shown to bind or interactwith CFTR include NHERF1 (Na+/H+ exchanger regulatory factor 1; alsoknown as EBP50), NHERF2 (Na+/H+ exchanger regulatory factor 2, alsoknown as E3KARP), NHERF3 (Na+/H+ exchanger regulatory factor 3, alsoknown as CAP70, PDZK1, or NaPi CAP-1), NHERF4 (Na+/H+ exchangerregulatory factor 4, also known as IKEPP or NaPi CAP-2), and CAL(CFTR-associated ligand; also known as PIST, GOPC, and FIG; GENBANKAccession Nos. NP_(—)065132 and NP_(—)001017408, incorporated herein byreference) (Guggino & Stanton (2006) supra; Li & Naren (2005) Pharmacol.Ther. 108:208-223). Of these proteins, CAL has been shown to reduce thelevels of recombinant wild-type CFTR found in whole cell lysates and atthe cell surface, whereas overexpression of NHERF1 together with CAL canblock this effect on both wild-type and ΔF508-CFTR (Cheng, et al. (2002)J. Biol. Chem. 277:3520-3529; Guerra, et al. (2005) J. Biol. Chem.280:40925-40933). Moreover, RNAi targeting of endogenous CALspecifically increases cell surface expression of the ΔF508-CFTR mutantprotein and enhances transepithelial chloride currents in a polarizedhuman patient bronchial epithelial cell line (Wolde, et al. (2007) J.Biol. Chem. 282:8099-8109). These data indicate that the PDZ proteinswhich interact with CFTR have opposing functions. Thus, targeting theinteraction of CAL with CFTR can stabilize a mutant CFTR protein andfacilitate cell surface expression of the same.

The CFTR protein and mutants thereof are well-known in the art andwild-type human CFTR is disclosed in GENBANK Accession No. NP_(—)000483,incorporated herein by reference. Misfolding of mutant CFTR proteins hasbeen shown to dramatically augment the ubiquitination susceptibility ofthe protein in post-Golgi compartments (Swiatecka-Urban, et al. (2005)J. Biol. Chem. 280:36762). Thus, for the purposes of the presentinvention, the term “degradation-prone” when used as a modifier of aCFTR protein, refers to a mutant CFTR protein that exhibits an increasedrate of degradation following initial trafficking to the cell surfaceand a decrease in the amount of CFTR protein present at the cell surface(i.e., plasma membrane). Examples of degradation-prone CFTR proteinsinclude, but are not limited to ΔF508 CFTR and Δ70F CFTR (see Sharma, etal. (2004) J. Cell Biol. 164:923). Other degradation-prone CFTR proteinsare known in the art and/or can be identified by routineexperimentation. For example, the rate or amount of transport of CFTRprotein from the cell surface can be determined by detecting the amountof complex-glycosylated CFTR protein present at the cell surface, inendoplasmic vesicles and/or in lysosomes using methods such as cellsurface immunoprecipitation or biotinylation or cell immunocytochemistrywith an antibody specific for CFTR protein. Additional methods, both invivo and in vitro, are known in the art that can be used for detectingan increase or decrease in cell surface expression of a CFTR protein.

Because PDZ proteins share overlapping specificities, particularembodiments of this invention embrace inhibitory agents that selectivelyblock the interaction or binding between a degradation-prone CFTR andCAL. As used herein, a “selective inhibitor of the CFTR and CALinteraction” or “an agent that selectively inhibits the interactionbetween the degradation-prone CFTR and CAL” is any molecular speciesthat is an inhibitor of the CFTR and CAL interaction but which fails toinhibit, or inhibits to a substantially lesser degree the interactionbetween CFTR and proteins that stabilize degradation-prone CFTR, e.g.,NHERF1 AND NHERF2. Methods for assessing the selectively of an inhibitorof the CFTR and CAL interaction are disclosed herein and can be carriedout in in vitro or in vivo assays.

By way of illustration, libraries of agents were screened for theability to increase the amount of ΔF508 CFTR at the apical membrane andto increase the CFTR-mediated chloride efflux across monolayers ofCFBE41O-cells. The magnitude of the functional rescue of the mutant CFTRprotein correlated with the selectivity of the agent for CAL versusNHERF1 and NHERF2, namely, the more selective the agent for the CALbinding site, the more effective the agent was at enhancing chlorideefflux.

Accordingly, the present invention features compositions and methods forfacilitating the cell surface expression of mutant CFTR by selectivelyblocking the interaction between a degradation-prone CFTR and CAL.Agents of the present invention can be any molecular species, withparticular embodiments embracing peptides or mimetics thereof.

As used herein, the term “peptide” denotes an amino acid polymer that iscomposed of at least two amino acids covalently linked by an amide bond.Peptides of the present invention are desirably 6 to 20 residues inlength, or more desirably 7 to 15 residues in length. In certainembodiments, a selective inhibitor of the CFTR and CAL interaction is a6 to 20 residue peptide containing the amino acid sequenceXaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Ile (SEQ ID NO:1), wherein Xaa₁ is Met, Phe orTrp; Xaa₂ is Gln, Pro, or Phe; Xaa₃ is Ser or Thr; Xaa₄ is Ser or Thr;and Xaa₅ is Lys or Ile. In certain embodiments of the present invention,a selective inhibitor of the CFTR and CAL interaction is a peptidehaving an amino acid sequence as listed in Table 1.

TABLE 1 Peptide Designation Peptide Sequence SEQ ID NO: PRC 01CANGLMQTSKI 2 PRC 02 CGLMQTSKI 3 PRC 03 CFFSTII 4 PRC 04 CFFTSII 5 PRC05 CMQTSII 6 PRC 06 CMQTSKI 7 PRC 07 CWQTSII 8 PRC 08 CWPTSII 9 PRC 09CTWQTSII 10 PRC 10 CKWQTSII 11 PRC 11 PHWQTSII 12 PRC 12 FHWQTSII 13 PRC13 SRWQTSII 14 PRC 17 CANSRWQTSII 15 PRC 25 GLWPTSII 16 PRC 26 SRWPTSII17 PRC 27 FPWPTSII 18 PRC 30 *FITC-ANSRWPTSII 19 PRC 36 ANSRWPTSII 20FITC = fluorescein.

In accordance with the present invention, derivatives of the peptides ofthe invention are also provided. As used herein, a peptide derivative isa molecule which retains the primary amino acids of the peptide,however, the N-terminus, C-terminus, and/or one or more of the sidechains of the amino acids therein have been chemically altered orderivatized. Such derivatized peptides include, for example, naturallyoccurring amino acid derivatives, for example, 4-hydroxyproline forproline, 5-hydroxylysine for lysine, homoserine for serine, ornithinefor lysine, and the like. Other derivatives or modifications include,e.g., a label, such as fluorescein or tetramethylrhodamine; or one ormore post-translational modifications such as acetylation, amidation,formylation, hydroxylation, methylation, phosphorylation, sulfatation,glycosylation, or lipidation. Indeed, certain chemical modifications, inparticular N-terminal glycosylation, have been shown to increase thestability of peptides in human serum (Powell et al. (1993) Pharma. Res.10:1268-1273). Peptide derivatives also include those with increasedmembrane permeability obtained by N-myristoylation (Brand, et al. (1996)Am. J. Physiol. Cell. Physiol. 270:C1362-C1369). An exemplary peptidederivative is provided in SEQ ID NO:19 (Table 1).

In addition, a peptide derivative of the invention can include acell-penetrating sequence which facilitates, enhances, or increases thetransmembrane transport or intracellular delivery of the peptide into acell. For example, a variety of proteins, including the HIV-1 Tattranscription factor, Drosophila Antennapedia transcription factor, aswell as the herpes simplex virus VP22 protein have been shown tofacilitate transport of proteins into the cell (Wadia and Dowdy (2002)Curr. Opin. Biotechnol. 13:52-56). Further, an arginine-rich peptide(Futaki (2002) Int. J. Pharm. 245:1-7), a polylysine peptide containingTat PTD (Hashida, et al. (2004) Br. J. Cancer 90(6):1252-8), Pep-1(Deshayes, et al. (2004) Biochemistry 43(6):1449-57) or an HSP70 proteinor fragment thereof (WO 00/31113) is suitable for enhancingintracellular delivery of a peptide or peptidomimetic of the inventioninto the cell. An exemplary cell penetrating peptide is shown in Table 2and provided as SEQ ID NO:30.

While a peptide of the invention can be derivatized with by one of theabove indicated modifications, it is understood that a peptide of thisinvention may contain more than one of the above described modificationswithin the same peptide.

As indicated, the present invention also encompasses peptidomimetics ofthe peptides disclosed herein. Peptidomimetics refer to a syntheticchemical compound which has substantially the same structural and/orfunctional characteristics of the peptides of the invention. The mimeticcan be entirely composed of synthetic, non-natural amino acid analogues,or can be a chimeric molecule including one or more natural peptideamino acids and one or more non-natural amino acid analogs. The mimeticcan also incorporate any number of natural amino acid conservativesubstitutions as long as such substitutions do not destroy the activityof the mimetic. Routine testing can be used to determine whether amimetic has the requisite activity, e.g., that it can inhibit theinteraction between CFTR and CAL. The phrase “substantially the same,”when used in reference to a mimetic or peptidomimetic, means that themimetic or peptidomimetic has one or more activities or functions of thereferenced molecule, e.g., selective inhibition of the CAL and CFTRinteraction.

There are clear advantages for using a mimetic of a given peptide. Forexample, there are considerable cost savings and improved patientcompliance associated with peptidomimetics, since they can beadministered orally compared with parenteral administration forpeptides. Furthermore, peptidomimetics are much cheaper to produce thanpeptides.

Thus, peptides described above have utility in the development of suchsmall chemical compounds with similar biological activities andtherefore with similar therapeutic utilities. The techniques ofdeveloping peptidomimetics are conventional. For example, peptide bondscan be replaced by non-peptide bonds or non-natural amino acids thatallow the peptidomimetic to adopt a similar structure, and thereforebiological activity, to the original peptide. Further modifications canalso be made by replacing chemical groups of the amino acids with otherchemical groups of similar structure. The development of peptidomimeticscan be aided by determining the tertiary structure of the originalpeptide, either free or bound to a CAL protein, by NMR spectroscopy,crystallography and/or computer-aided molecular modeling. Thesetechniques aid in the development of novel compositions of higherpotency and/or greater bioavailability and/or greater stability than theoriginal peptide (Dean (1994) BioEssays 16:683-687; Cohen & Shatzmiller(1993) J. Mol. Graph. 11:166-173; Wiley & Rich (1993) Med. Res. Rev.13:327-384; Moore (1994) Trends Pharmacol. Sci. 15:124-129; Hruby (1993)Biopolymers 33:1073-1082; Bugg, et al. (1993) Sci. Am. 269:92-98). Oncea potential peptidomimetic compound is identified, it may be synthesizedand assayed using an assay described herein or any other appropriateassay for monitoring cell surface expression of CFTR.

It will be readily apparent to one skilled in the art that apeptidomimetic can be generated from any of the peptides describedherein. It will furthermore be apparent that the peptidomimetics of thisinvention can be further used for the development of even more potentnon-peptidic compounds, in addition to their utility as therapeuticcompounds.

Peptide mimetic compositions can contain any combination of non-naturalstructural components, which are typically from three structural groups:residue linkage groups other than the natural amide bond (“peptidebond”) linkages; non-natural residues in place of naturally occurringamino acid residues; residues which induce secondary structural mimicry,i.e., induce or stabilize a secondary structure, e.g., a beta turn,gamma turn, beta sheet, alpha helix conformation, and the like; or otherchanges which confer resistance to proteolysis. For example, apolypeptide can be characterized as a mimetic when one or more of theresidues are joined by chemical means other than an amide bond.Individual peptidomimetic residues can be joined by amide bonds,non-natural and non-amide chemical bonds other chemical bonds orcoupling means including, for example, glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropyl-carbodiimide(DIC). Linking groups alternative to the amide bond include, forexample, ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, 7:267-357, “Peptide and BackboneModifications,” Marcel Decker, N.Y.).

As discussed, a peptide can be characterized as a mimetic by containingone or more non-natural residues in place of a naturally occurring aminoacid residue. Non-natural residues are known in the art. Particularnon-limiting examples of non-natural residues useful as mimetics ofnatural amino acid residues are mimetics of aromatic amino acidsinclude, for example, D- or L-naphylalanine; D- or L-phenylglycine; D-or L-2 thieneylalanine; D- or L-1, -2,3-, or 4-pyreneylalanine; D- orL-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- orL-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- orL-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine;D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- orL-p-biphenylphenylalanine; D- or L-p-methoxy-biphenyl-phenylalanine; andD- or L-2-indole(alkyl)alanines, where alkyl can be substituted orunsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl,iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acid. Aromaticrings of a non-natural amino acid that can be used in place a naturalaromatic ring include, for example, thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Cyclic peptides or cyclized residue side chains also decreasesusceptibility of a peptide to proteolysis by exopeptidases orendopeptidases. Thus, certain embodiments embrace a peptidomimetic ofthe peptides disclosed herein, whereby one or more amino acid residueside chains are cyclized according to conventional methods.

Mimetics of acidic amino acids can be generated by substitution withnon-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; and sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) including, for example,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl groups can also be converted to asparaginyl and glutaminylgroups by reaction with ammonium ions.

Lysine mimetics can be generated (and amino terminal residues can bealtered) by reacting lysinyl with succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate.

Methionine mimetics can be generated by reaction with methioninesulfoxide. Proline mimetics of include, for example, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- or 4-methylproline, and3,3,-dimethylproline.

One or more residues can also be replaced by an amino acid (orpeptidomimetic residue) of the opposite chirality. Thus, any amino acidnaturally occurring in the L-configuration (which can also be referredto as R or S, depending upon the structure of the chemical entity) canbe replaced with the same amino acid or a mimetic, but of the oppositechirality, referred to as the D-amino acid, but which can additionallybe referred to as the R- or S-form.

As will be appreciated by one skilled in the art, the peptidomimetics ofthe present invention can also include one or more of the modificationsdescribed herein for derivatized peptides, e.g., a label, one or morepost-translational modifications, or cell-penetrating sequence.

As with peptides of the invention, peptidomimetics are desirably 6 to 20residues in length, or more desirably to 15 residues in length. Incertain embodiments, a selective inhibitor of the CFTR and CALinteraction is a 6 to 20 residue peptidomimetic based on the amino acidsequence of SEQ ID NO:1. In certain embodiments of the presentinvention, a selective inhibitor of the CFTR and CAL interaction is apeptidomimetic listed in Table 2.

TABLE 2 Peptide Designation Peptide Sequence SEQ ID NO: PRC 21WrFK(K-FITC)-ANSRWPTSII 21 PRC 23 WrFKK-ANSRWPTSII 22 PRC 29WrFK(K-ROX)-ANSRWPTSII 23 PRC 37 pneaWPTSII 24 B1 fNaRWQTSII 25 B2fNSRWQTSII 26 B3 knSRWQTSII 27 B4 pnSRWQTSII 28 A6 AnSRWQTSII 29Lower-case = D-amino acids; FITC = fluorescein; ROX =6-carboxy-X-rhodamine. Underlined residues indicate cyclized sidechains. WrFKK (SEQ ID NO: 30) is a cell penetrating peptide.

Also included with the scope of the invention are peptides andpeptidomimetics that are substantially identical to a sequence set forthherein, in particular SEQ ID NO:1. The term “substantially identical,”when used in reference to a peptide or peptidomimetic, means that thesequence has at least 75% or more identity to a reference sequence(e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%). The length of comparisonsequences will generally be at least 5 amino acids, but typically more,at least 6 to 10, 7 to 15, or 8 to 20 residues. In one aspect, theidentity is over a defined sequence region, e.g., the amino or carboxyterminal 3 to 5 residues.

The peptides, derivatives and peptidomimetics can be produced andisolated using any method known in the art. Peptides can be synthesized,whole or in part, using chemical methods known in the art (see, e.g.,Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980)Nucleic Acids Res. Symp. Ser. 225-232; and Banga (1995) TherapeuticPeptides and Proteins, Formulation, Processing and Delivery Systems,Technomic Publishing Co., Lancaster, Pa.). Peptide synthesis can beperformed using various solid-phase techniques (see, e.g., Roberge(1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) andautomated synthesis may be achieved, e.g., using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the manufacturer'sinstructions.

Individual synthetic residues and peptides incorporating mimetics can besynthesized using a variety of procedures and methodologies known in theart (see, e.g., Organic Syntheses Collective Volumes, Gilman, et al.(Eds) John Wiley & Sons, Inc., NY). Peptides and peptide mimetics canalso be synthesized using combinatorial methodologies. Techniques forgenerating peptide and peptidomimetic libraries are well-known, andinclude, for example, multipin, tea bag, and split-couple-mix techniques(see, for example, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby(1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers.3:17-27; and Ostresh (1996) Methods Enzymol. 267:220-234). Modifiedpeptides can be further produced by chemical modification methods (see,for example, Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel(1995) Free Radic. Biol. Med. 19:373-380; and Blommers (1994)Biochemistry 33:7886-7896).

Alternatively, peptides of this invention can be prepared in recombinantprotein systems using polynucleotide sequences encoding the peptides. Byway of illustration, a nucleic acid molecule encoding a peptide of theinvention is introduced into a host cell, such as bacteria, yeast ormammalian cell, under conditions suitable for expression of the peptide,and the peptide is purified or isolated using methods known in the art.See, e.g., Deutscher et al. (1990) Guide to Protein Purification:Methods in Enzymology Vol. 182, Academic Press.

It is contemplated that the peptides and mimetics disclosed herein canbe used as lead compounds for the design and synthesis of compounds withimproved efficacy, clearance, half-lives, and the like. One approachincludes structure-activity relationship (SAR) analysis (e.g., NMRanalysis) to determine specific binding interactions between the agentand CAL or CFTR to facilitate the development of more efficaciousagents. Agents identified in such SAR analysis or from agent librariescan then be screened for their ability to increase cell surfaceexpression of CFTR.

In this regard, the present invention also relates to a method foridentifying an agent for which facilitates cell surface expression of adegradation-prone CFTR. The method of the invention involves contactingCAL with a test agent under conditions allowing an interaction betweenthe agent and CAL, and determining whether the agent competitivelydisplaces binding of a degradation-prone CFTR to CAL. Particulardegradation-prone CFTRs that can be used include, but are not limitedto, ΔF508 and R1066C.

In one embodiment, the method is performed in vivo. Various detectionmethods can be employed to determine whether the agent displaces CFTRfrom CAL. For example, displacement can be based on detecting anincrease in an amount of CFTR protein on the cell surface,immunostaining with a specific antibody (e.g., anti-CFTR, M3A7), ordirect visualization (e.g., a CFTR-GFP fusion). Additional methodsuseful for determining whether there is an increase in cell surfaceprotein included cell panning. In cell panning assays, plates are coatedwith an antibody that binds to the cell surface protein. The number ofcells that binds to the antibody coated plate corresponds to an amountof protein on the cell surface.

In another embodiment, the method is performed in vitro. In accordancewith this embodiment, a combination of peptide-array screening andfluorescence polarization is used to identify agents that bind to anisolated, recombinant CAL PZD domain. For example, it contemplated thatthe high-affinity CAL-binding peptides disclosed herein can be use asreporters for small-molecule screening assays, wherein the smallmolecules compete for binding to the CAL PZD domain. The ability totarget PDZ proteins selectively, using a combination of peptide-arrayscreening and fluorescence-polarization assays on purified, recombinantPDZ domains, represents a novel achievement, due to the bi-directionalpromiscuity of PDZ:protein interactions. Since PDZ proteins areimplicated in the trafficking and intracellular localization of manydisease-related receptors, selective targeting may provide an importanttool for identifying additional PDZ-based therapeutics.

In so far as it is desirable that the agent selectively inhibit theinteraction between CAL and CFTR, a further embodiment of this inventionembraces contacting NHERF1 and/or NHERF2 with an identified inhibitor ofthe CAL and CFTR interaction and determining whether the agentcompetitively displaces binding to NHERF1 and/or NHERF2. Agents thatfail to inhibit, or inhibit to a substantially lesser degree theinteraction between CFTR and NHERF1 or NHERF2 as compared to CAL, wouldbe considered selective.

Agents which can be screened in accordance with the methods disclosedherein can be from any chemical class including peptides, antibodies,small organic molecules, carbohydrates, etc.

Agents specifically disclosed herein, as well as derivatives, andpeptidomimetics of said agents and agents identified by design and/orscreening assays find application in increasing in the cell surfaceexpression of degradation-prone CFTR proteins and in the treatment ofCF. Thus, methods for increasing the cell surface expression of adegradation-prone CFTR and treating cystic fibrosis are also provided bythis invention.

In accordance with one embodiment, the cell surface expression of adegradation-prone CFTR protein is enhanced or increased by contacting acell expressing a degradation-prone CFTR with an agent that decreases orinhibits the interaction between the CFTR protein and CAL so that thecell surface expression of the CFTR protein is increased or enhanced.Desirably, the agent is administered in an amount that effectivelystabilizes the degradation-prone CFTR protein and increases the amountof said CFTR protein present or detectable at the cell surface by atleast 60%, 70%, 80%, 90%, 95%, 99% or 100% as compared to cells notcontacted with the agent. Any cell can be employed in this method of theinvention so long as it expresses a degradation-prone CFTR. Specificexamples of such cells include, but are not limited to, primary cells ofa subject with CF or cultured airway epithelial cell lines derived froma CF patient's bronchial epithelium (e.g., CFBE41O-). It is contemplatedthat this method of the invention can be used to increase cell surfaceexpression of a degradation-prone CFTR protein in a human subject aswell as increase the cell surface expression of a degradation-prone CFTRprotein in an isolated cell or cell culture to, e.g., study thetransport and/or activity of the mutant protein at the cell surface.

In another embodiment, a subject with CF or at risk of CF is treatedwith one or more the agents of the invention. In accordance with thisembodiment, an effective amount of an agent that selectively inhibitsthe interaction between a degradation-prone CFTR and CAL is administeredto a subject in need of treatment thereby preventing or treating thesubject's cystic fibrosis. Subjects benefiting from treatment with anagent of the invention include subjects confirmed as having CF, subjectssuspected of having CF, or subjects at risk of having CF (e.g., subjectswith a family history). In one aspect, the subject expresses adegradation-prone CFTR, such as ΔF508 or R1066C CFTR. Other CFTR mutantsequences are also known in the art including, for example, ΔI507,N1303K, S549I, S549R, A559T, H139R, G149R, D192G, R258G, S949L, H949Y,H1054D, G1061R, L1065P, R1066C, R1066H, R1066L, Q1071P, L 1077P, H1085R,W1098R, M1101K, M1101R.

Successful clinical use of a selective inhibitor of the invention can bedetermined by the skilled clinician based upon routine clinicalpractice, e.g., by monitoring frequency of respiratory infections and/orcoughing; or changes in breathing, abdominal pain, appetite, and/orgrowth according to methods known in the art.

Agents disclosed herein can be employed as isolated molecules (i.e.,isolated peptides, derivatives, or peptidomimetics), or in the case ofpeptides, be expressed from nucleic acids encoding said peptides. Suchnucleic acids can, if desired, be naked or be in a carrier suitable forpassing through a cell membrane (e.g., DNA-liposome complex), containedin a vector (e.g., plasmid, retroviral vector, lentiviral, adenoviral oradeno-associated viral vectors and the like), or linked to inert beadsor other heterologous domains (e.g., antibodies, biotin, streptavidin,lectins, etc.), or other appropriate compositions. Thus, both viral andnon-viral means of nucleic acid delivery can be achieved and arecontemplated. Desirably, a vector used in accordance with the inventionprovides all the necessary control sequences to facilitate expression ofthe peptide. Such expression control sequences can include but are notlimited to promoter sequences, enhancer sequences, etc. Such expressioncontrol sequences, vectors and the like are well-known and routinelyemployed by those skilled in the art.

For example, when using adenovirus expression vectors, the nucleic acidmolecule encoding a peptide can be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. Alternatively, the vaccinia virus 7.5Kpromoter can be used. (see e.g., Mackett, et al. (1982) Proc. Natl.Acad. Sci. USA 79:7415-7419; Mackett, et al. (1984) J. Virol.49:857-864; Panicali, et al. (1982) Proc. Natl. Acad. Sci. USA79:4927-4931). Mammalian expression systems further include vectorsspecifically designed for “gene therapy” methods including adenoviralvectors (U.S. Pat. Nos. 5,700,470 and 5,731,172), adeno-associatedvectors (U.S. Pat. No. 5,604,090), herpes simplex virus vectors (U.S.Pat. No. 5,501,979) and retroviral vectors (U.S. Pat. Nos. 5,624,820,5,693,508 and 5,674,703 and WIPO publications WO 92/05266 and WO92/14829).

Moreover, agents of the invention can be combined with other agentsemployed in the treatment of CF, including molecules which amelioratethe signs or symptoms of CF. Such agents include, but are not limitedto, nonsteroidal anti-inflammatory drugs or steroids, such as ibuprofenfor treating inflammation; pentoxifylline for decreasing inflammation;dornase alfa for treating airway blockage due to mucus buildup orcertain flavones and isoflavones, which are capable of stimulatingCFTR-mediated chloride transport in epithelial tissues in a cyclic-AMPindependent manner (U.S. Pat. No. 6,329,422); 2,2-dimethyl butyric acid(U.S. Pat. No. 7,265,153); glycerol, acetic acid, butyric acid, D- orL-amino-n-butyric acid, alpha- or beta-amino-n-butyric acid, argininebutyrate or isobutyramide, all disclosed in U.S. Pat. Nos. 4,822,821 and5,025,029; butyrin, 4-phenyl butyrate, phenylacetate, and phenoxy aceticacid, disclosed in U.S. Pat. No. 4,704,402, wherein in combination withone or more agents of this invention, an additive or synergistic effectis achieved.

For therapeutic use, agents of the invention (including nucleic acidsencoding peptides) can be formulated with a pharmaceutically acceptablecarrier at an appropriate dose. Such pharmaceutical compositions can beprepared by methods and contain carriers which are well-known in theart. A generally recognized compendium of such methods and ingredientsis Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro,editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000.A pharmaceutically acceptable carrier, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, is involved in carrying or transporting the subject agent fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be acceptable in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient.

Examples of materials which can serve as pharmaceutically acceptablecarriers include sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose, and its derivatives,such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients, such ascocoa butter and suppository waxes; oils, such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Compositions of the present invention can be administered parenterally(for example, by intravenous, intraperitoneal, subcutaneous orintramuscular injection), topically including via inhalation,transdermally, orally, intranasally, intravaginally, or rectallyaccording to standard medical practices.

The selected dosage level of an agent will depend upon a variety offactors including the activity of the particular agent of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularagent being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particular agentemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and other factorswell-known in the medical arts.

A physician having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired based upon the administration of similar compounds orexperimental determination. For example, the physician could start dosesof an agent at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. This is considered to be within the skill ofthe artisan and one can review the existing literature on a specificagent or similar agents to determine optimal dosing.

The fact that other proteins destined for the intracellular transportpathway frequently exhibit transport delays due to mutations, or otherfactors, indicates that the cell-surface expression of suchdegradation-prone proteins may also be mediated by CAL. Thus, it iscontemplated that the agents of this invention can also be used toinduce or increase the cell surface expression of otherdegradation-prone proteins. Accordingly, physiological disordersassociated with other degradation-prone proteins besides CFTR cansimilarly be treated using the methods disclosed herein. Physiologicaldisorders associated with a degradation-prone protein that can betreated in a method of the invention include, for example, Stargardt'sdisease and particular types of macular dystrophy caused by mutations ofthe retinal rod transporter, ABC-R, resulting in deficiency of export.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Identification of Selective Inhibitors of the CAL and CFTRInteraction

Using peptide-array screening and fluorescence-polarization bindingassays, a series of peptide sequences were identified that bind CALprogressively more tightly than CAL binds to CFTR, and that in parallelbind NHERF1 and NHERF2 progressively more weakly than these proteinsbind to CFTR.

To test the ability of CAL inhibitors to rescue CFTR, cultured airwayepithelial cells (cell line CFBE41o-, derived from a CF patient'sBronchial Epithelium) were grown on filters, permitting formation ofpolarized cell monolayers similar to those found in epithelial tissues.

The CFBE41o-cell line is well-recognized as an airway epithelial modelsystem for the study of CF processes. These cells express the mostcommon disease mutant associated with CF, ΔF508-CFTR, which ischaracterized by the loss of a single amino acid codon at position 508of CFTR. Roughly 50% of CF patients are homozygous for ΔF508-CFTR, andanother 40% are heterozygotes for this allele. Functional rescue ofΔF508-CFTR therefore has the potential to alleviate symptoms in up to90% of CF patients. Although very little ΔF508-CFTR protein issynthesized in the absence of intervention, the protein itself retainssome functional activity. If rescued and stabilized it can restorephysiological CFTR activity, potentially reversing the processes thatlead to chronic lung infection, and ultimately death, in most CFpatients.

When introduced into CFBE41o-cells using commercial peptide transfectionreagents, representative peptide and peptidomimetic compounds were ableto increase the amount of ΔF508-CFTR protein at the apical membrane andto increase the CFTR-mediated chloride efflux across the monolayers. Themagnitude of the functional rescue correlated with the selectivity ofthe peptides for CAL vs. NHERF1 and NHERF2; the more selective thepeptide for the CAL binding site, the more effective it was at enhancingchloride efflux.

Furthermore, when used in combination with a compound that enhances thebiosynthesis of ΔF508-CFTR (a “corrector”), the instant inhibitorsshowed an additive effect, comparable in magnitude to that of thecorrector compound.

Although compounds have previously been designed to enhance thesynthesis and/or chloride-channel activity of CFTR, the instantinhibitors were designed to stabilize mutant CFTR protein that hasalready been synthesized within the cell and successfully transported tothe cell surface. The peptides and peptidomimetics disclosed hereinprovide a basis for further optimization of CAL inhibitor properties interms of affinity and selectivity for CAL, in vivo proteolyticstability, cellular uptake, and ADME characteristics.

EXAMPLE 2 Assays for Assessing Activity of Selective Inhibitors

Agents of the present invention can be assayed for their ability tostimulate chloride transport in epithelial tissues. Such transport mayresult in secretion or absorption of chloride ions. The ability tostimulate chloride transport may be assessed using any of a variety ofsystems. For example, in vitro assays using a mammalian trachea or acell line, such as the permanent airway cell line Calu-3 (ATCC AccessionNumber HTB55) may be employed. Alternatively, the ability to stimulatechloride transport may be evaluated within an in vivo assay employing amammalian nasal epithelium. In general, the ability to stimulatechloride transport may be assessed by evaluating CFTR-mediated currentsacross a membrane by employing standard Ussing chamber (see Ussing &Zehrahn (1951) Acta. Physiol. Scand. 23:110-127) or nasal potentialdifference measurements (see Knowles, et al. (1995) Hum. Gene Therapy6:445-455). Within such assays, an agent that stimulates a statisticallysignificant increase in chloride transport at a concentration of about1-300 μM is said to stimulate chloride transport.

Within one in vitro assay, the level of chloride transport may beevaluated using mammalian pulmonary cell lines, such as Calu-3 cells, orprimary bovine tracheal cultures. In general, such assays employ cellmonolayers, which may be prepared by standard cell culture techniques.Within such systems, CFTR-mediated chloride current may be monitored inan Ussing chamber using intact epithelia. Alternatively, chloridetransport may be evaluated using epithelial tissue in which thebasolateral membrane is permeabilized with Staphylococcus aureusα-toxin, and in which a chloride gradient is imposed across the apicalmembrane (see Illek, et al. (1996) Am. J. Physiol. 270:C265-75). Ineither system, chloride transport is evaluated in the presence andabsence of a test agent, and those compounds that stimulate chloride maybe used within the methods provided herein.

Within another in vitro assay for evaluating chloride transport, cells,such as NIH 3T3 fibroblasts, are transfected with a CFTR gene having amutation associated with cystic fibrosis (e.g., ΔF508-CFTR) using wellknown techniques (see Anderson, et al. (1991) Science 25:679-682). Theeffect of an agent on chloride transport in such cells is then evaluatedby monitoring CFTR-mediated currents using the patch clamp method (seeHamill, et al. (1981) Pflugers Arch. 391:85-100) with and without agent.

Alternatively, such assays may be performed using a mammalian trachea,such as a primary cow tracheal epithelium using the Ussing chambertechnique as described above. Such assays are performed in the presenceand absence of a test agent to identify agents that stimulate chloridetransport.

What is claimed is:
 1. An isolated peptide comprisingXaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-lle (SEQ ID NO: 1), wherein Xaa₁ is Met or Trp;Xaa₂ is Gln, Pro, or Phe; Xaa₃ is Ser or Thr; Xaa₄ is Ser or Thr; andXaa₅ is Lys or Ile, or a derivative or peptidomimetic thereof whereinthe peptide, derivative or peptidomimetic is 6 to 20 residues in length.2. An isolated peptide consisting of the sequence selected from thegroup of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
 3. Anisolated peptidomimetic consisting of the sequence selected from thegroup of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.
 4. Apharmaceutical composition comprising the peptide of claim 1 inadmixture with a pharmaceutically acceptable carrier.
 5. Apharmaceutical composition comprising the peptide of claim 2 inadmixture with a pharmaceutically acceptable carrier.
 6. Apharmaceutical composition comprising the peptidomimetic of claim 3 inadmixture with a pharmaceutically acceptable carrier.